In the medical and veterinary clinical setting, detection and species identification of harmful bacteria infecting biological fluids or tissues is a pre-requisite for appropriate and timely relevant antibiotherapy. Such identification is classically performed by conventional microbiological methods (culture on solid medium or in liquid phase). These conventional methods have however their own limitations.
Culture is always followed by phenotypic identification, which is based on the biochemical features of the bacteria. Usually, the whole process requires 48 to 72 hours to be completed. This period is unfortunately too long, considering the speed of bacterial growth in infected tissues and, for some bacteria, the pathological effects related the toxins that they produce. This time is also too long when bacteria are spread in the environment (aerosol, food or water contamination), where germs are able to infect humans or animals and spread rapidly on a epidemic way from an infected to a healthy body on a very short time. There is therefore a need for the rapid detection and identification of pathogenic bacterial agent(s) involved in human or animal infections or present in the environment.
A stream of studies carried out recently has confirmed that molecular identification is more efficient than phenotypic identification (Bosshard et al, 2003; Bosshard et al, 2004: Lecouvet et al, 2004) and genotypic definition of bacteria species has now become the gold standard (Clarridge, 2004). There is therefore an increasing need for identifying bacterial species with more reliable methods. While obvious in the hospital setting, it is also of interest of the post September 2001 era, where accuracy and speed in identification of deadly bacteria are priorities.
Aside of the time required for routine microbiologic detection, another limiting factor is sometimes the lack of bacterial growth, generating a false-negative microbiologic result. False-negative bacterial cultures are not unusual in the clinical practice, even when clinical and biological signs clearly suggest a florid and active infection (Lecouvet et al, 2004). This false-negativity may be due to a low organism burden, non-culturable or slowly growing micro-organisms or, most often, to prior antibiotic therapy (Trampuz et al, 2003; Tzanakaki et al, 2003). In this case, a false-negative result hampers correct etiological diagnosis regarding the bacterial origin of the infectious disease, and precludes the use of early targeted antibiotherapy. As delayed antibiotherapy may increase the risk of worse clinical outcome (Gutierrez et al, 1998; Yu et al, 2003, Lecouvet et al, 2004), this situation often prompts the use of empiric, broad spectrum and sometimes long-term therapy, and certainly when there is no microbiologic result.
The higher sensitivity, speed and accuracy of DNA amplification by PCR for identification of bacteria is expected to reduce the time to diagnosis, to improve the diagnostic rate, and to allow an early choice of specific antibiotic treatment. Over the last decade, this expectation has fuelled the development of numerous promising DNA assays for detecting and identifying bacteria at the species- or genera-level in human and environmental samples (Jonas et al, 2003; Palomares et al, 2003; Poyart et al, 2001; Xu et al, 2002).
These assays remain however restricted to single species and/or genera (Brakstad et al, 1992; Poyart et al, 2001; Vannuffel et al, 1998). Such restriction has various disadvantages. For instance, in the absence of any indication on the presence of bacterial agents in an environmental sample or in a biological tissue/fluid sample from human or animal origin suspected to be infected but showing no bacterial background due to the presence of a normal bacterial flora, molecular screening methods have to be applied which target the greatest as possible number of potentially pathogenic bacteria including the most feared bacteria (Staphylococci, Streptococci, Bacillus anthracis, Enterobacteriacea, Neisseria, etc. . . . ) that could be used by bioterrorists. In this case, the use of specific markers or well-defined genera requires multiple and/or repeated testing to confirm or exclude a bacterial diagnosis. Considering the cost of this strategy as well as the limited amount DNA usually available for one sample, this is practically impossible to be performed.
In another example, in samples from tissues showing a bacterial background due the presence of a normal flora, the identification of a well defined panel of pathogenic bacteria recognized as “prior key targets” in the clinical setting considered (e.g. community-acquired pneumonia) remains very difficult.
In view of the above, there is therefore a need for the rapid detection and identification of pathogenic bacterial agent(s) involved in human or animal infections or present in the environment.
There is also a need for identification and diagnostic tools, which allow screening for the presence of pathogenic bacterial agent(s), and to detect and identify these pathogenic bacteria within a bacterial background.
In particular, it is clear that there is a great need in the art for molecular screening/detection and identification assays and methods having a range of specificity that is as wide as possible in order to quickly detect the presence of bacteria (bacterial detection step), while allowing in parallel or subsequently, to identify the present bacterial species, genera and, optionally the strain (bacterial identification step).
In a first aspect, the present invention therefore aims to provide an improved assay for detecting micro-organisms, and in particular bacteria. It is further an aim of the invention to provide an improved assay for diagnosing bacterial infection of a sample and/or tissue.
In another aspect, the present invention also aims to provide an improved assay and method for the identification of micro-organisms. More in particular, the invention aims to identify and provide a series of specific, molecular markers for the detection and/or identification of micro-organisms, and preferably bacteria, in a Gram-, genus- species- and/or strain-specific way.